The specific aims of the proposed research are to characterize the structural, vibronic, and electronic properties of the photophysically active bacteriochlorophyll (BChl) and bacteriopheophytin (BPh) cofactors in bacterial photosynthetic reaction centers (RCs). Bacterial RCs were chosen for study because of this class of proteins represents a prototypical model system for investigating the factors which mediate charge separation across a biological membrane. The utility of bacterial RCs for examining this process is due to the fact that (1) high-resolution X-ray crystallographic data are available for RCs from several species and (2) genetic engineering techniques are well developed for certain of these species. These two criteria are not satisfied for any mammalian membrane-bound proteins involved in primary charge-separation and energy-transduction processes. The principal investigative tool for the studies is resonance Raman (RR) spectroscopy. The first major objective is to characterize the properties of the BChl/BPh cofactors in genetically modified RCs which exhibit novel electron-transfer properties. These studies will focus on a series of mutants of Rb, capsulatus wherein replacements have been made in amino acid residues which are in close proximity to the accessory BChl and BPh cofactors on the electron-transfer active L branch of the protein. The genetically modified RCs will include mutants in which electron transfer has been first documented to proceed down the normally inactive M branch of the protein. In all cases the RR studies will be conducted on RCs hose detailed electron-transfer kinetics have been elucidated via time-resolved optical experiments. The second major objective is to conduct RR studies on RCs aimed at determining the nature of the low-frequency vibrational modes of athe BChl/BPh cofactors which are strongly coupled to the lowest- energy optical transition(s). The principal target of these studies is the primary electron donor. The characterization of the low-frequency modes will proceed via acquisition and analysis of RR data from RCs labeled with 15N, 26Mg, and 15N/26Mg. The long-term objective of all the studies is to determine how the physical properties of the cofactors (structure, electron-density distribution, electron-phonon coupling) govern and/or reflect their functional characteristics (electron transfer and charge separation across the biological membrane).